The paradigm that mangrove carbon supports secondary production in mangrove and adjacent habitats has been debated in recent years. Fatty acids (FA) are one of the classic biomarkers that have been frequently applied to track mangrove carbon pathways and assess trophic relationships. However, most previous studies did not evaluate the validity, potential and limitations of FA as biomarkers. The function and metabolism of long-chain polyunsaturated FA (LC-PUFA) in mangrove fauna have been largely ignored, and overlapping single FA biomarkers were widely used to infer dietary contributions from different sources. This review aims to systematically analyze and assess the application of FA biomarkers to dietary analyses in mangrove ecosystems, with a focus on basal food sources and their consumers. Our results show that basal food sources have distinctive FA profiles, with leaves and litter rich in alpha-linolenic acids (ALA, 18:3n-3), microphytobenthos rich in eicosapentaenoic acids (EPA, 20:5n-3) and suspended particulate organic matter (SPOM), or phytoplankton rich in docosahexaenoic acids (DHA, 22:6n-3). Most consumers contain high contents of LC-PUFA, particularly DHA and EPA, but very low levels of long-chain saturated FA (e.g., 22:0, 24:0, 26:0, 28:0), a biomarker of mangrove leaf litter. Bacterial FA biomarkers are present in all consumers. Four possible carbon pathways are identified and examined, i.e., benthic feeding on mangrove leaves and litter, benthic feeding on microphytobenthos, pelagic feeding on SPOM, and benthic and pelagic feeding on bacteria. Each pathway plays a different nutritional role for consumers, together providing a diversity of carbon sources. We recommend that in future (a) a wide range of basal diet sources should be sampled rather than just “visible” sources; (b) the unique FA characteristics of each diet source and consumer should be recognized with a focus on overall FA profiles and the application of multivariate statistics; (c) controlled feeding trials should be considered for keystone or functionally important consumers before selecting certain FA biomarkers to infer animal diets, and; (d) compound-specific stable isotope analysis should be applied to provide more insights into trophic relationships as well as the FA metabolic pathways in consumers.